Comparative Label-Free Proteomics Study on Celiac Disease-Active Epitopes in Common Wheat, Spelt, Durum Wheat, Emmer, and Einkorn

Wheat species with various ploidy levels may be different regarding their immunoreactive potential in celiac disease (CD), but a comprehensive comparison of peptide sequences with known epitopes is missing. Thus, we used an untargeted liquid chromatography tandem mass spectrometry method to analyze the content of peptides with CD-active epitope in the five wheat species common wheat, spelt, durum wheat, emmer, and einkorn. In total, 494 peptides with CD-active epitope were identified. Considering the average of the eight cultivars of each species, spelt contained the highest number of different peptides with CD-active epitope (193 ± 12, mean ± SD). Einkorn showed the smallest variability of peptides (63 ± 4) but higher amounts of certain peptides compared to the other species. The wheat species differ in the presence and distribution of CD-active epitopes; hence, the entirety of peptides with CD-active epitope is crucial for the assessment of their immunoreactive potential.


Overview
Table S1.Overview of samples: Species, cultivar and abbreviation.
Table S2.Protein database: Triticum proteins (UniProtKB) with at least one celiac disease active epitope (according Sollid et al. 2019) as of 23.03.2021 (please see separate excel file).
Table S3.Identified peptides with celiac disease-active epitope highlighting the epitope (please see separate excel file).
Table S4.Number of cultivars per species, in which the peptides with celiac disease-active epitope were identified (please see separate excel file).
Table S5.Significance of components of principle component analysis of LFQ intensities of peptides with celiac disease active epitope based on the species.Table S6.Statistical parameters of principle component analysis of LFQ intensities of peptides with celiac disease active epitope based on the species.Text S1.Additional description of Figure 6.
Regarding the presence of EPeps with epitope FS (Figure 6), the samples showed no cluster by species or ploidy level.The samples RAM (emmer) and BAS (spelt) did not contain any of the EPeps.Column cluster 1 and 2 contained samples of all species whereas column cluster 3 only contained spelt, durum wheat and emmer samples and column cluster 4 contained the majority of common wheat samples, two spelt samples and one durum wheat sample.Row cluster A contained EPeps mainly present in samples of column cluster 4. EPeps of row cluster B were present in single samples but not in samples of column cluster 3. EPeps of row cluster C were present in all samples of column cluster 2 and in single samples of column cluster 1 and 4. Row cluster D contained EPeps present in the majority of samples in column cluster 1, 2 and 3.
Text S2.Additional description of Figure 7.
Row cluster A summarized EPeps present in the cluster of durum wheat and emmer (column cluster 1) and the cluster of spelt (column cluster 2).EPeps of row cluster B were present in the majority of the samples of column cluster 1 and single samples of column cluster 2 (spelt samples and common wheat sample MUL).In both row clusters, one EPep each was identified additionally in the emmer sample OSI.EPeps of row cluster C were present in the majority of hexaploid samples, but not in tetraploid and diploid samples.EPeps of row cluster D were present in the majority of common wheat samples.
One EPep was additionally present in the spelt sample BAS and another EPep in the emmer samples of column cluster 5. Row cluster E summarized EPeps present in single samples.
Text S3.Additional description of Figure S2.
EPeps that were present in the majority of the einkorn samples were grouped in row cluster A. Only one EPep of this cluster was also present in some hexaploid samples.Row cluster B contained EPeps present in hexaploid samples and single tetraploid samples.The EPeps of row cluster C were present in samples of all species.Row cluster D contained EPeps present in all emmer samples of column cluster 1, and in one emmer sample of column clusters 3 and 7, respectively.Row cluster E summarized EPeps present in single samples of all species.The EPeps of row cluster F were present in the majority of the samples of column cluster 3 and in some samples of column cluster 4 and 7. Row cluster G contained EPeps present in all samples of column cluster 3 and 4 as well as in some samples of column cluster 5, 6 and 7. Row cluster H showed EPeps present in the hexaploid samples besides the ones of column cluster 6.The EPeps of row cluster I were present in all species except einkorn.

Figure S1 .
Figure S1.Distribution of the number of identified peptides with celiac disease active epitope according to their length.

Figure S2 .
Figure S2.Intensity based on label-free quantitation of identified peptides with celiac disease active epitope QGYYPTSPQ belonging to proteins of the high-molecular-weight-glutenin subunits in common wheat, spelt, durum wheat, emmer and einkorn samples.

Figure S3 .
Figure S3.Intensity based on label-free quantitation of identified peptides with celiac disease active epitopes FSQQQQSPF, PFSQQQQPV and QGYYPTSPQ belonging to glutenin proteins in common wheat, spelt, durum wheat, emmer and einkorn samples.

Figure S4 .
Figure S4.Intensity based on label-free quantitation of identified peptides with celiac disease active epitopes FRPQQPYPQ and QYSQPQQPI (epitope group 1) belonging to α-gliadin proteins in common wheat, spelt, durum wheat, emmer and einkorn samples.

Figure S5 .
Figure S5.Intensity based on label-free quantitation of identified peptides with celiac disease active epitopes QGSFQPSQQ and QGSVQPQQL (epitope group 2) belonging to α-gliadin proteins in common wheat, spelt, durum wheat, emmer and einkorn samples.

Figure S6 .
Figure S6.Intensity based on label-free quantitation of identified peptides with celiac disease active epitopes PYPQPQLPY, PFPQPQLPY and PQPQLPYPQ (epitope group 3) belonging to α-gliadin proteins in common wheat, spelt, durum wheat, emmer and einkorn samples.

Figure S7 .
Figure S7.Intensity based on label-free quantitation of identified peptides with celiac disease active epitopes PQQSFPQQ, SQPQQQFPQ and PQPQQQFPQ (epitope group 4) belonging to γ-gliadin proteins in common wheat, spelt, durum wheat, emmer and einkorn samples.

Figure S1 .
Figure S1.Distribution of the number of identified peptides with celiac disease active epitope according to their length between eight and 46 amino acids.

Figure S2 .
Figure S2.Intensity based on label-free quantitation of identified peptides with celiac disease active epitope QGYYPTSPQ belonging to proteins of the highmolecular-weight-glutenin subunits in common wheat, spelt, durum wheat, emmer and einkorn samples.

Figure S3 .
Figure S3.Intensity based on label-free quantitation of identified peptides with celiac disease active epitopes FSQQQQSPF, PFSQQQQPV and QGYYPTSPQ belonging to glutenin proteins in common wheat, spelt, durum wheat, emmer and einkorn samples.

Figure S4 .
Figure S4.Intensity based on label-free quantitation of identified peptides with celiac disease active epitopes FRPQQPYPQ and QYSQPQQPI (epitope group 1) belonging to α-gliadin proteins in common wheat, spelt, durum wheat, emmer and einkorn samples.

Figure S5 .
Figure S5.Intensity based on label-free quantitation of identified peptides with celiac disease active epitopes QGSFQPSQQ and QGSVQPQQL (epitope group 2) belonging to α-gliadin proteins in common wheat, spelt, durum wheat, emmer and einkorn samples.

Figure S6 .
Figure S6.Intensity based on label-free quantitation of identified peptides with celiac disease active epitopes PYPQPQLPY, PFPQPQLPY and PQPQLPYPQ (epitope group 3) belonging to α-gliadin proteins in common wheat, spelt, durum wheat, emmer and einkorn samples, 33-mer is indicated by an asterisk.

Figure S7 .
Figure S7.Intensity based on label-free quantitation of identified peptides with celiac disease active epitopes PQQSFPQQQ, SQPQQQFPQ and PQPQQQFPQ (epitope group 4) belonging to γ-gliadin proteins in common wheat, spelt, durum wheat, emmer and einkorn samples.

Table S1 .
Overview of samples: species, cultivar and abbreviation.

Table S2 .
Protein database: Triticum proteins (UniProtKB) with at least one celiac disease active epitope (according Sollid et al. 2019) as of 23.03.2021 (please see separate excel file).

Table S3 .
Identified peptides with celiac disease active epitope highlighting the containing epitope (please see separate excel file).

Table S4 .
Number of cultivars per species, in which the peptides with celiac disease active epitope were identified (please see separate excel file).

Table S5 .
Significance of components of principle component analysis of LFQ intensities of peptides with celiac disease active epitope based on the species.

Table S6 .
Statistical parameters of principle component analysis of LFQ intensities of peptides with celiac disease active epitope based on the species.